Drug Therapy for Celiac Sprue

ABSTRACT

Administering an effective dose of a tTGase inhibitor to a Celiac or dermatitis herpetiformis patient reduces the toxic effects of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten.

BACKGROUND OF THE INVENTION

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes a disease calledCeliac Sprue in sensitive individuals. Gluten is a complex mixture ofglutamine- and proline-rich glutenin and prolamine molecules and isthought to be responsible for induction of Celiac Sprue. Ingestion ofsuch proteins by sensitive individuals produces flattening of thenormally luxurious, rug-like, epithelial lining of the small intestineknown to be responsible for efficient and extensive terminal digestionof peptides and other nutrients. Other clinical symptoms of Celiac Sprueinclude fatigue, chronic diarrhea, malabsorption of nutrients, weightloss, abdominal distension, anemia, as well as a substantially enhancedrisk for the development of osteoporosis and intestinal malignanciessuch as lymphoma and carcinoma. The disease has an incidence ofapproximately 1 in 200 in European populations and is believed to besignificantly under diagnosed in other populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption of the skin characterized by clusters of intensely pruriticvesicles, papules, and urticaria-like lesions. IgA deposits occur inalmost all normal-appearing and perilesional skin. Asymptomaticgluten-sensitive enteropathy is found in 75 to 90% of patients and insome of their relatives. Onset is usually gradual. Itching and burningare severe, and scratching often obscures the primary lesions witheczematization of nearby skin, leading to an erroneous diagnosis ofeczema. Strict adherence to a gluten-free diet for prolonged periods maycontrol the disease in some patients, obviating or reducing therequirement for drug therapy. Dapsone, sulfapyridine, and colchicinesare sometimes prescribed for relief of itching.

Celiac Sprue (CS) is generally considered to be an autoimmune diseaseand the antibodies found in the serum of the patients support the theorythat the disease is immunological in nature. Antibodies to tissuetransglutaminase (tTGase or tTG) and gliadin appear in almost 100% ofthe patients with active CS, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villous atrophy of the small intestine.

At the present time, there is no good therapy for the disease, except toavoid completely all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. A leading cause of death islymphoreticular disease, especially intestinal lymphoma. It is not knownwhether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for example, in commercial soups, sauces, icecreams, hot dogs, and other foodstuffs, that patients need detailedlists of foodstuffs to avoid and expert advice from a dietitian familiarwith celiac disease. Ingesting even small amounts of gluten may preventremission or induce relapse. Supplementary vitamins, minerals, andhematinics may also be required, depending on deficiency. A few patientsrespond poorly or not at all to gluten withdrawal, either because thediagnosis is incorrect or because the disease is refractory. In thelatter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) mayinduce response.

In view of the serious and widespread nature of Celiac Sprue and thedifficulty of removing gluten from the diet, better methods of treatmentare of great interest. In particular, there is a need for treatmentmethods that allow the Celiac Sprue individual to eat gluten-containingfoodstuffs without ill effect or at least to tolerate such foodstuffs insmall or moderate quantities without inducing relapse. The presentinvention meets this need for better therapies for Celiac Sprue byproviding new drugs and methods and formulations of new and existingdrugs to treat Celiac Sprue. International Patent ApplicationUS03/04743, herein specifically incorporated by reference, disclosesaspects of gluten protease stability and immunogenicity.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treatingCeliac Sprue and/or dermatitis herpetiformis and the symptoms thereof byadministration of a tTGase (tissue transglutaminase) inhibitor to thepatient. In one embodiment, the tTGase inhibitor employed in the methodis a small molecule tTGase inhibitor comprising a3-halo-4,5-dihydroisoxazole derivative, as described herein. In anotheraspect, the present invention provides novel derivative compounds of3-halo-4,5-dihydroisoxazoles. In one embodiment, the tTGase inhibitoremployed in the method is an analog of isatin (2, 3 diketoindoline).

In another aspect, the invention provides pharmaceutical formulationscomprising a tTGase inhibitor and a pharmaceutically acceptable carrier.In one embodiment, the formulation also comprises one or moreglutenases, as described in International Patent Application WO03/068170. The invention also provides methods for the administration ofenteric formulations of one or more of the tTGase inhibitors of thepresent invention to treat Celiac Sprue. In another aspect, the tTGaseinhibitors and/or pharmaceutical formulations of the present inventionare useful in treating disorders where TGases are a factor in thedisease etiology, where such disorders may include cancer, neurologicaldisorders, wound healing, etc. These conditions include Alzheimer's andHuntington's diseases, where the TGases appear to be a factor in theformation of inappropriate proteinaceous aggregates that may becytotoxic. In diseases such as progressive supranuclear palsy,Huntington's, Alzheimer's and Parkinson's diseases, the aberrantactivation of TGases may be caused by oxidative stress and inflammation.

These and other aspects and embodiments of the invention and methods formaking and using the invention are described in more detail in thedescription of the drawings and the invention, the examples, the claims,and the drawings that follow.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are treated by inhibitionof tissue transglutaminase with specific inhibitors as described herein.Methods and compositions are provided for the administration of one ormore tTGase inhibitors to a patient suffering from Celiac Sprue and/ordermatitis herpetiformis. The compositions of the invention includeformulations of tTGase inhibitors that comprise an enteric coating thatallows delivery of the agents to the intestine in an active form; theagents are stabilized to resist digestion or alternative chemicaltransformations in acidic stomach conditions. In another embodiment,food is pretreated or combined with glutenase, or a glutenase isco-administered (whether in time or in a formulation of the invention)with a tTGase inhibitor of the invention The subject methods are usefulfor both prophylactic and therapeutic purposes. Thus, as used herein,the term “treating” is used to refer to both prevention of disease, andtreatment of a pre-existing condition. The treatment of ongoing disease,to stabilize or improve the clinical symptoms of the patient, is aparticularly important benefit provided by the present invention. Suchtreatment is desirably performed prior to loss of function in theaffected tissues; consequently, the prophylactic therapeutic benefitsprovided by the invention are also important. Evidence of therapeuticeffect may be any diminution in the severity of disease, particularlydiminution of the severity of such symptoms as fatigue, chronicdiarrhea, malabsorption of nutrients, weight loss, abdominal distension,and anemia. Other disease indicia include the presence of antibodiesspecific for glutens, antibodies specific for tissue transglutaminase,the presence of pro-inflammatory T cells and cytokines, and degradationof the villus structure of the small intestine. Application of themethods and compositions of the invention can result in the improvementof any and all of these disease indicia of Celiac Sprue.

Patients that can benefit from the present invention include both adultsand children. Children in particular benefit from prophylactictreatment, as prevention of early exposure to toxic gluten peptides canprevent development of the disease into its more severe forms. Childrensuitable for prophylaxis in accordance with the methods of the inventioncan be identified by genetic testing for predisposition, e.g. by HLAtyping; by family history, and by other methods known in the art. As isknown in the art for other medications, and in accordance with theteachings herein, dosages of the tTGase inhibitors of the invention canbe adjusted for pediatric use.

Compounds of interest for inhibition of tTGase include those having thegeneral formulae

where R₁, R₂ and R₃ are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups. R₁ and R₂ can also be an amino acid, apeptide, a peptidomimetic, or a peptidic protecting groups.

Illustrative R₁ groups include Cbz, Fmoc, and Boc. In other embodimentsof the invention, R₁ is an arylether, aryl, alkylether or alkyl group,e.g. O-benzyl, benzyl, methyl or ethyl.

R₂ groups of interest include OMe, OtBu, Gly, and Gly-NH₂. In otherembodiments,

R₂ is selected from the group consisting of (s)-Bn, (s)-CO₂Me, (s)-Me,(R)-Bn, (S)—CH₂CONHBn, (S)-(1H-inol-yl)-methyl, and(S)-(4-hydrohy-phenyl)-methyl.

R₃ is preferably a halo group, i.e. F, Cl, Br, and I. In someembodiments of the invention R₃ is a halogen other than Cl, i.e.selected from the group consisting of I, F and Br.

X₁ and X₂ are selected from the group consisting of NH, O, and NR₄,where R₄ is a lower alkyl.

n is a whole number between 0 and 10, usually between 0 and 5, and moreusually between 0 and 3.

The tTGase inhibitory compounds of the invention from the isoxazoles canbe readily prepared using methods known in the art for other purposesand the teachings herein. Examples of synthetic routes to thesecompounds are also described in examples below For example, Castelhanoet al have demonstrated that the dihydroisoxazole derivative(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}carbamicacid benzyl ester is an inhibitor of bovine epidermal transglutaminase(Castelhano et al., Bioorg. Chem. (1988) 16, 335-340). The followinggeneral formula for transglutaminase inhibitors is disclosed inEP0237082:

Here we identify new compounds within this genus that are especiallyeffective inhibitors of human tissue transglutaminase, and may thereforebe used to treat Celiac Sprue and/or Dermatitis Herpetiforms. We alsodisclose new compounds with comparable activity.

Another example of tTGase inhibitors are analogs of the dioxoindolineisatin. The cyclic α-keto amide structure of isatin serves as a goodanalog of γ-carboxamide group of tTGase glutamyl substrate. α-ketoamides are widely utilized as reversible inhibitors ofcysteine-dependent proteases and, in a similar way, the hetetocyclicstructure of isatin possesses an electrophilic carbonyl group whichcould be recognized by the enzyme as an analog of the substrateγ-carboxamide carbonyl group. Using standard procedures known in theart, the aromatic portion of the isatin structure can be derivatizedfurther to incorporate additional functional groups into the inhibitorsmimicking the other parts of peptide substrates.

Isatin analogs of interest may have the following general formula:

where R₁, R₂ and R₃ are may be the same or different, and areindependently selected from H, a halo group, i.e. F, Cl, Br, and I,alkyls, including lower alkyls, aryls, and NO₂.

Certain compounds of interest as tTGase inhibitors are set forth inTable I:

TABLE 1 Dissociation Constants (K_(l)) and Inhibition Rate Constants(k_(inh)) of Dihydroisoxazole Inhibitors of Human tTGase K_(l) k_(inh)k_(inh)/K_(l) (mM) (min⁻¹) (min⁻¹M⁻¹)

1a R = CH₂C₆H₅ 0.74 1.3 1900  b CH₂C₆H₄-p-OH 0.42 0.86 2000  cCH₂C₆H₄-p-F 0.43 0.42 980  d CH₂C₆H₄-m-F 0.39 0.84 2200  e CH₂-3-indolyl0.31 0.78 2500  f CH₂-3-(5-OH-indolyl) 0.11 0.31 2800  g CH₂CONHCH₂Ph0.24 0.54 2300  h (R)-CH₂Ph 0.31 0.29 940  i CH₃ 0.91 0.41 450

2a X = O 1.3 0.32 230  b NCH₃ 0.26 0.19 730

3a X = H, R = Me 2.7 0.60 220  b X = OH, R = O-4-picolyl 0.081 0.12 1500 c O-3-picolyl 0.078 0.21 2700  d OCH₂CH₂Ph 0.061 0.093 1500  eO-2-naphthyl 0.043 0.070 1600  f

0.087 0.38 4400

4 R = CH₂-3-(5-OH-indolyl) 0.079 0.54 6800The illustrative compounds of the invention described above were testedin a tTGase assay with recombinant human tissue transglutaminase, whichwas expressed, purified and assayed as described (Piper et al.,Biochemistry (2001) 41, 386-393). Competitive inhibition with respect tothe Cbz-Gln-Gly substrate was observed for all substrates; in all casesirreversible inactivation of the enzyme was also observed.

To facilitate an appreciation of the invention, the tTGase inhibitors ofthe invention have in part been described above with structurescontaining variable “R” groups that are defined by reference to thevarious organic moieties that can be present at the indicated positionin the structure. Below, brief definitions are provided for the phrasesused to define the organic moieties listed for each R group.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain radical consisting solely of carbon and hydrogen atoms, containingno unsaturation, having from one to eight carbon atoms, and which isattached to the rest of the molecule by a single bond, e.g., methyl,ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), and the like. Unless stated otherwisespecifically in the specification, the alkyl radical may be optionallysubstituted by hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl. Unless stated otherwise specificallyin the specification, it is understood that for radicals, as definedbelow, that contain a substituted alkyl group that the substitution canoccur on any carbon of the alkyl group.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy (isopropoxy), n-butoxy, n-pentoxy, 1,1-dimethylethoxy(t-butoxy), and the like. Unless stated otherwise specifically in thespecification, it is understood that for radicals, as defined below,that contain a substituted alkoxy group that the substitution can occuron any carbon of the alkoxy group. The alkyl radical in the alkoxyradical may be optionally substituted as described above.

“Alkylthio” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above, e.g., methylthio, ethylthio,n-propylthio, 1-methylethylthio (iso-propylthio), n-butylthio,n-pentylthio, 1,1-dimethylethylthio (t-butylthio), and the like. Unlessstated otherwise specifically in the specification, it is understoodthat for radicals, as defined below, that contain a substitutedalkylthio group that the substitution can occur on any carbon of thealkylthio group. The alkyl radical in the alkylthio radical may beoptionally substituted as described above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing at least onedouble bond, having from two to eight carbon atoms, and which isattached to the rest of the molecule by a single bond or a double bond,e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl,and the like. Unless stated otherwise specifically in the specification,the alkenyl radical may be optionally substituted by hydroxy, alkoxy,haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂,—C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)—C(O)—R⁸ where each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.Unless stated otherwise specifically in the specification, it isunderstood that for radicals, as defined below, that contain asubstituted alkenyl group that the substitution can occur on any carbonof the alkenyl group.

“Aryl” refers to a phenyl or naphthyl radical. Unless stated otherwisespecifically in the specification, the term “aryl” or the prefix “ar-”(such as in “aralkyl”) is meant to include aryl radicals optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or—N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl. The term “aryl” alsorefers to the compound C₈H₅, i.e. Bn.

“Aralkyl” refers to a radical of the formula —R_(a)R_(b) where R_(a) isan alkyl radical as defined above and R_(b) is one or more aryl radicalsas defined above, e.g., benzyl, diphenylmethyl and the like. The arylradical(s) may be optionally substituted as described above.

“Aralkenyl” refers to a radical of the formula —R_(c)R_(b) where R_(c)is an alkenyl radical as defined above and R_(b) is one or more arylradicals as defined above, e.g., 3-phenylprop-1-enyl, and the like. Thearyl radical(s) and the alkenyl radical may be optionally substituted asdescribed above.

“Alkylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain may beoptionally substituted by one or more substituents selected from thegroup consisting of aryl, halo, hydroxy, alkoxy, haloalkoxy, cyano,nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂or —N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl. The alkylene chain may beattached to the rest of the molecule through any two carbons within thechain.

“Alkenylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing at least onedouble bond and having from two to eight carbon atoms, e.g., ethenylene,prop-1-enylene, but-1-enylene, pent-1-enylene, hexa-1,4-dienylene, andthe like. The alkenylene chain may be optionally substituted by one ormore substituents selected from the group consisting of aryl, halo,hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio,cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸ where each R⁸is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl,aralkyl or aryl. The alkenylene chain may be attached to the rest of themolecule through any two carbons within the chain.

“Cycloalkyl” refers to a stable monovalent monocyclic or bicyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,having from three to ten carbon atoms, and which is saturated andattached to the rest of the molecule by a single bond, e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and thelike. Unless otherwise stated specifically in the specification, theterm “cycloalkyl” is meant to include cycloalkyl radicals which areoptionally substituted by one or more substituents independentlyselected from the group consisting, of alkyl, aryl, aralkyl, halo,haloalkyl, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto,alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl.

“Cycloalkylalkyl” refers to a radical of the formula —R_(a)R_(d) whereR_(a) is an alkyl radical as defined above and R_(d) is a cycloalkylradical as defined above. The alkyl radical and the cycloalkyl radicalmay be optionally substituted as defined above.

“Halo” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Haloalkoxy” refers to a radical of the formula —OR_(c) where R_(c) isan haloalkyl radical as defined above, e.g., trifluoromethoxy,difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy,1-fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy,1-bromomethyl-2-bromoethoxy, and the like.

“Heterocyclyl” refers to a stable 3- to 15-membered ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. For purposes ofthis invention, the heterocyclyl radical may be a monocyclic, bicyclicor tricyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be aromatic or partiallyor fully saturated. The heterocyclyl radical may not be attached to therest of the molecule at any heteroatom atom. Examples of suchheterocyclyl radicals include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzothiadiazolyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, carbazolyl, cinnolinyl,decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl, isothiazolyl,imidazolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, indolyl,indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isoxazolyl,isoxazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolyl,oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl,quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl,thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl,tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl sulfoxide,and thiamorpholinyl sulfone. Unless stated otherwise specifically in thespecification, the term “heterocyclyl” is meant to include heterocyclylradicals as defined above which are optionally substituted by one ormore substituents selected from the group consisting of alkyl, halo,nitro, cyano, haloalkyl, haloalkoxy, aryl, heterocyclyl,heterocyclylalkyl, —OR⁸, —R⁷—OR⁸, —C(O)OR⁸, —R⁷—C(O)OR⁸, —C(O)N(R⁸)₂,—N(R⁸)₂, —R⁷—N(R⁸)₂, and —N(R⁸)C(O)R⁸ wherein each R⁷ is a straight orbranched alkylene or alkenylene chain and each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.

“Heterocyclylalkyl” refers to a radical of the formula —R_(a)R_(e) whereR_(a) is an alkyl radical as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. The heterocyclyl radical may beoptionally substituted as defined above.

In the formulas provided herein, molecular variations are included,which may be based on isosteric replacement. “Isosteric replacement”refers to the concept of modifying chemicals through the replacement ofsingle atoms or entire functional groups with alternatives that havesimilar size, shape and electro-magnetic properties, e.g. 0 is theisosteric replacement of S, N, COOH is the isosteric replacement oftetrazole, F is the isosteric replacement of H, sulfonate is theisosteric replacement of phosphate etc.

As used herein, compounds which are “commercially available” may beobtained from standard commercial sources including Acros Organics(Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., including SigmaChemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

As used herein, “suitable conditions” for carrying out a synthetic stepare explicitly provided herein or may be discerned by reference topublications directed to methods used in synthetic organic chemistry.The reference books and treatise set forth above that detail thesynthesis of reactants useful in the preparation of compounds of thepresent invention, will also provide suitable conditions for carryingout a synthetic step according to the present invention.

As used herein, “methods known to one of ordinary skill in the art” maybe identified though various reference books and databases. Suitablereference books and treatise that detail the synthesis of reactantsuseful in the preparation of compounds of the present invention, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specificand analogous reactants may also be identified through the indices ofknown chemicals prepared by the Chemical Abstract Service of theAmerican Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., www.acs.org may be contacted formore details). Chemicals that are known but not commercially availablein catalogs may be prepared by custom chemical synthesis houses, wheremany of the standard chemical supply houses (e.g., those listed above)provide custom synthesis services.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

The tTGase inhibitors, or their pharmaceutically acceptable salts maycontain one or more asymmetric centers and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as, their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, such as reverse phase HPLC. Whenthe compounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included.

The present invention provides the tTGase inhibitors in a variety offormulations for therapeutic administration. In one aspect, the agentsare formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and areformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the tTGase inhibitors is achievedin various ways, although oral administration is a preferred route ofadministration. In some formulations, the tTGase inhibitors are systemicafter administration; in others, the inhibitor is localized by virtue ofthe formulation, such as the use of an implant that acts to retain theactive dose at the site of implantation.

In some pharmaceutical dosage forms, the tTGase inhibitors areadministered in the form of their pharmaceutically acceptable salts. Insome dosage forms, the tTGase inhibitor is used alone, while in others,the tTGase is used in combination with another pharmaceutically activecompounds. In the latter embodiment, the other active compound is, insome embodiments, a glutenase that can cleave or otherwise degrade atoxic gluten oligopeptide, as described in the Examples below. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the agents are used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and in some embodiments, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, and cellulose acetate phthalate.

Other enteric formulations of the tTGase inhibitors of the inventioncomprise engineered polymer microspheres made of biologically erodablepolymers, which display strong adhesive interactions withgastrointestinal mucus and cellular linings, can traverse both themucosal absorptive epithelium and the follicle-associated epitheliumcovering the lymphoid tissue of Peyer's patches. The polymers maintaincontact with intestinal epithelium for extended periods of time andactually penetrate it, through and between cells. See, for example,Mathiowitz et al. (1997) Nature 386 (6623): 410-414. Drug deliverysystems can also utilize a core of superporous hydrogels (SPH) and SPHcomposite (SPHC), as described by Dorkoosh et al. (2001) J ControlRelease 71(3):307-18.

In another embodiment, the tTGase inhibitor or formulation thereof isadmixed with food, or used to pre-treat foodstuffs containing glutens.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of tTGase inhibitor calculated in an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular complexemployed and the effect to be achieved, and the pharmacodynamicsassociated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Depending on the patient and condition being treated and on theadministration route, the tTGase inhibitor is administered in dosages of0.01 mg to 500 mg V/kg body weight per day, e.g. about 100 mg/day for anaverage person. Dosages are appropriately adjusted for pediatricformulation. Those of skill will readily appreciate that dose levels canvary as a function of the specific inhibitor, the diet of the patientand the gluten content of the diet, the severity of the symptoms, andthe susceptibility of the subject to side effects. Some of theinhibitors of the invention are more potent than others. Preferreddosages for a given inhibitor are readily determinable by those of skillin the art by a variety of means. A preferred means is to measure thephysiological potency of a given compound.

The methods of the invention are useful in the treatment of individualssuffering from Celiac Sprue and/or dermatitis herpetiformis, byadministering an effective dose of a tTGase inhibitor, through apharmaceutical formulation, and the like. Diagnosis of suitable patientsmay utilize a variety of criteria known to those of skill in the art. Aquantitative increase in antibodies specific for gliadin, and/or tissuetransglutaminase is indicative of the disease. Family histories and thepresence of the HLA alleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8[DQ(a1*0301, b1*0302)] are indicative of a susceptibility to thedisease. Moreover, as tTG plays an important role in other diseases,such as Huntington's disease and skin diseases in addition to dermatitisherpetiformis, a variety of formulated versions of the compounds of theinvention (e.g. topical formulations, intravenous injections) are usefulfor the treatment of such medical conditions. These conditions includeAlzheimer's and Huntington's diseases, where the TGases appear to be afactor in the formation of inappropriate proteinaceous aggregates thatmay be cytotoxic. In diseases such as progressive supranuclear palsy,Huntington's, Alzheimer's and Parkinson's diseases, the aberrantactivation of TGases may be caused by oxidative stress and inflammation.

Therapeutic effect is measured in terms of clinical outcome, or byimmunological or biochemical tests. Suppression of the deleteriousT-cell activity can be measured by enumeration of reactive Th1 cells, byquantitating the release of cytokines at the sites of lesions, or usingother assays for the presence of autoimmune T cells known in the art.Also both the physician and patient can identify a reduction in symptomsof a disease.

Various methods for administration are employed in the practice of theinvention. In one preferred embodiment, oral administration, for examplewith meals, is employed. The dosage of the therapeutic formulation canvary widely, depending upon the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the patient, and the like. The initial dose can be larger, followedby smaller maintenance doses. The dose can be administered asinfrequently as weekly or biweekly, or more often fractionated intosmaller doses and administered daily, with meals, semi-weekly, and thelike, to maintain an effective dosage level.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature), but someexperimental errors and deviations may be present. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Synthesis of Dihydroxyisoxazole Containing tTGase Inhibitors

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 1A, withreference to Formula 5, n=0, X=NH, R₁=BnO, R₂=(S)-Bn, R₃=Br).N-Cbz-L-Phe (0.30 g, 1.0 mmol) and HOBt (0.15 g, 1.1 eq) were dissolvedin 2 mL DMF. 3-Bromo-5-aminomethyl-4,5-dihydroisoazole (0.18 g, 1.0 eq),prepared following a reported procedure (Rohloff et al., (1992)Tetrahedron Lett 33(22):3113-3116), was added to the solution cooled inan ice bath followed by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (0.23 g, 1.2 eq). The ice bath was removed and thestirring was continued overnight. The solution was diluted with ethylacetate and washed with sat. NaHCO₃ solution and brine. The organiclayer was dried over MgSO₄ and filtered. The solvent was removed byevaporation and the residue was purified by SiO₂ chromatography to givethe title compound as a white solid (0.24 g, 52%).

¹H NMR (CDCl₃, 200 MHz): δ=7.34-7.26 (m, 8H), 7.17 (d, 2H, J=7.6 Hz),6.19-6.09 (m, 1H), 5.21-5.15 (m, 1H), 5.09 (s, 2H), 4.74-4.60 (m, 1H),4.41-4.36 (m, 1H), 3.49-3.45 (m, 2H), 3.26-3.12 (m, 1H), 3.07 (d, 2H,J=6.8 Hz), 2.97-2.76 (m, 1H)

MS (ESI): m/z=460.1 [M+H]⁺, 482.2 [M+Na]⁺

Synthesis of(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-butyricacid methyl ester (with reference to Formula 5, n=2, X=NH, R₁=BnO,R₂=(S)—CO₂Me, R₃=Br) (50). The title compound was prepared according tothe procedure for compound 1A except using N-Cbz-L-Glu-OMe.

¹H NMR (CDCl₃, 200 MHz): δ=7.41-7.30 (m, 5H), 6.22-6.12 (m, 1H),5.63-5.57 (m, 1H), 5.11 (s, 2H), 4.82-4.74 (m, 1H), 4.41-4.33 (m, 1H),3.75 (s, 3H), 3.54-3.48 (m, 2H), 3.32-3.15 (m, 1H), 3.02-2.88 (m, 1H),2.34-2.22 (m, 3H), 2.05-1.94 (m, 1H)

MS (ESI): m/z=456.1 [M+H]⁺, 478.2 [M+Na]⁺

Synthesis of(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-succinamicacid methyl ester (with reference to Formula 5, n=1, X=NH, R₁=BnO,R₂=(S)—CO₂Me, R₃=Br) (51). The title compound was prepared according tothe procedure for compound 1A except using N-Cbz-L-Asp-OMe.

¹H NMR (CDCl₃, 200 MHz): δ=7.37-7.30 (m, 5H), 6.00-5.90 (m, 2H), 5.13(s, 2H), 4.80-4.71 (m, 1H), 4.63-4.58 (m, 1H), 3.76 (s, 3H), 3.54-3.44(m, 2H), 3.33-3.23 (m, 1H), 2.99-2.70 (m, 3H)

MS (ESI): m/z=442.1 [M+H]⁺, 464.2 [M+Na]⁺

Synthesis of (S)-2-Benzyloxycarbonylamino-3-phenyl-propionic acid3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester (with reference to Formula5, n=0, X=O, R₁=BnO, R₂=(S)—Bn, R₃=Br) (With reference to Table 1, 2a).N-Cbz-L-Phe (0.30 g, 1.0 mmol) was dissolved in the mixture ofacetonitrile (6 mL), DIEA (0.18 mL, 1.0 eq) and excess allyl bromide (3mL). After the reaction was allowed to proceed overnight, the reactionmixture was diluted with ethyl acetate, washed with sat. Na₂CO₃ solutionand brine, dried over MgSO₄ and concentrated to provide the allyl esteras a clear oil (0.34 g, quant.). The ester (0.19 g, 0.57 mmol) anddibromoformaldoxime (0.14 g, 1.1 eq) were dissolved in 3 mL ethylacetated and NaHCO₃ (0.21 g, 4.3 eq) was added to the solution. Thereaction mixture was stirred overnight, diluted with ethyl acetated andwashed with sat. NaHCO₃ solution and brine. The organic layer was driedover MgSO₄ and the solvent was removed by evaporation. The residue waspurified by SiO₂ chromatography to give the title compound as a whitesolid (0.15 g, 58%)

¹H NMR (CDCl₃, 200 MHz): δ=7.35-7.26 (m, 8H), 7.16-7.14 (m, 2H),5.20-5.05 (m, 3H), 4.85-4.79 (m, 1H), 4.68-4.63 (m, 1H), 4.22-4.15 (m,2H), 3.27-3.09 (m, 3H), 2.96-2.77 (m, 1H)

MS (ESI): m/z=461.1 [M+H]⁺, 483.2 [M+Na]⁺

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamicacid benzyl ester (With reference to Table 1, Structure 1i; withreference to Formula 5, n=0, X=NH, R₁=BnO, R₂=(S)-Me, R₃=Br). The titlecompound was prepared according to the procedure for compound 1A exceptusing N-Cbz-L-Ala.

¹H NMR (CDCl₃, 200 MHz): δ=7.37-7.34 (b, 5H), 6.68-6.45 (m, 1H),5.24-5.18 (m, 1H), 5.13 (s, 2H), 4.80-4.76 (m, 1H), 4.26-4.18 (m, 1H),3.55-3.47 (m, 2H), 3.33-3.19 (m, 1H), 3.05-2.92 (m, 1H), 1.39 (d, 3H,J=7.0 Hz)

MS (ESI): m/z=384.1 [M+H]⁺, 406.1 [M+Na]⁺

Synthesis of(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-propionamide(With reference to Table 1, structure 3a, with reference to Formula 5,n=0, X=NH, R₁=Me, R₂=(S)-Bn, R₃=Br). The title compound was preparedaccording to the procedure for compound 1A except using N—Ac-L-Phe.

¹H NMR (CDCl₃, 200 MHz): δ=7.33-7.18 (m, 5H), 6.14-6.09 (m, 1H),6.02-5.97 (m, 1H), 4.67-4.59 (m, 2H), 3.49-3.41 (m, 2H), 3.22-3.03 (m,3H), 2.97-2.70 (m; 1H), 2.00 (s, 3H)

MS (ESI): m/z=368.1 [M+H]⁺, 390.2 [M+Na]⁺

Synthesis of{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (With reference to Table 1, Structure 1 h, withreference to Formula 5, n=0, X=NH, R₁=BnO, R₂=(R)-Bn, R₃=Br). The titlecompound was prepared according to the procedure for compound 1A exceptusing N-Cbz-D-Phe.

¹H NMR (CDCl₃, 200 MHz): δ=7.34-7.26 (m, 8H), 7.17 (d, 2H, J=7.8 Hz),6.19-6.09 (m, 1H), 5.21-5.15 (m, 1H), 5.09 (s, 2H), 4.74-4.60 (m, 1H),4.41-4.36 (m, 1H), 3.49-3.45 (m, 2H), 3.26-3.12 (m, 1H), 3.07 (d, 2H,J=7.0 Hz), 2.97-2.76 (m, 1H)

MS (ESI): m/z=460.1 [M+H]⁺, 482.2 [M+Na]⁺

Synthesis of{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 1g, withreference to Formula 5, n=0, X=NH, R₁=BnO, R₂=(S)—CH₂CONHBn, R₃=Br). Thetitle compound was prepared according to the procedure for compound 1Aexcept using β-benzylamide of N-Cbz-L-Asp((S)—N-Benzyl-2-benzyloxycarbonylamino-succinamic acid).

¹H NMR (CDCl₃, 400 MHz): δ=7.38-7.24 (m, 1H), 6.43-6.40 (m, 1H),6.01-5.99 (m, 1H), 5.14 (s, 2H), 4.80-4.70 (m, 1H), 4.58-4.52 (m, 1H),4.41 (d, 2H, J=6.4 Hz), 3.57-3.50 (m, 2H), 3.25-3.12 (m, 1H), 3.00-3.94(m, 2H), 2.62-2.56 (m, 1H)

MS (ESI): m/z 517.1 [M+H]⁺, 539.2 [M+Na]⁺

Synthesis of[(S)-1-[(3-Bromo-4,5-dihydroisoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl]-carbamicacid benzyl ester (With reference to Formula 5, n=0, X=NH, R₁=BnO,R₂=(S)-(1H-indol-3-yl)-methyl, R₃=Br) (57). The title compound wasprepared according to the procedure for compound 1A except usingN-Cbz-L-Trp.

¹H NMR (CDCl₃, 400 MHz): δ=8.14 (br, 1H), 7.70-7.63 (m, 1H), 7.37-7.31(m, 6H), 7.22-7.18 (m, 1H), 7.13-7.09 (m, 1H), 7.04-7.02 (m, 1H),6.15-6.10 (m, 1H), 5.45-5.39 (m, 1H), 5.14-5.06 (m, 2H), 4.59-4.47 (m,2H), 3.40-3.31 (m, 3H), 3.20-3.14 (m, 1H), 3.11-3.04 (m, 1H), 2.82-2.74(m, 1H)

MS (ESI): m/z=499.0 [M+H]⁺, 521.2 [M+Na]⁺

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 2b, withreference to Formula 5, n=0, X=NMe, R₁=BnO, R₂=(S)-Bn, R₃=Br). The titlecompound was prepared according to the procedure for compound 2a exceptusing N-methylallylamine.

¹H NMR (CDCl₃, 400 MHz): δ=7.34-7.26 (m, 8H), 7.18-7.16 (m, 2H),5.57-5.56 (m, 1H), 5.12-5.05 (m, 2H), 4.93-4.73 (m, 2H), 3.80-3.67 (m,1H), 3.36-3.17 (m, 2H), 3.02-3.86 (m, 6H)

MS (ESI): m/z=474.2 [M+H]⁺, 496.3 [M+Na]⁺

Synthesis of[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamicacid benzyl ester (With reference to Formula 5, n=0, X=NH, R₁=BnO,R₂=(S)-(4-hydroxy-phenyl)-methyl, R₃=Br) (59). The title compound wasprepared according to the procedure for compound 1a except usingN-Cbz-L-Tyr.

¹H NMR (DMSO-d₆, 400 MHz): δ=9.17 (br, 1H), 8.27-8.23 (m, 1H), 7.43-7.40(m, 1H), 7.32-7.22 (m, 5H), 7.03 (d, 2H, J=7.6 Hz), 6.62 (d, 2H, J=7.6Hz), 4.93 (s, 2H), 4.68-4.64 (m, 1H), 4.13-4.11 (m, 1H), 3.37-3.19 (m,3H), 3.05-2.90 (m, 1H), 2.81-2.77 (m, 1H), 2.63-2.58 (m, 1H)

MS (ESI): m/z=476.1 [M+H]⁺, 498.2 [M+Na]⁺

Synthesis of 1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-urea(With reference to Formula 5, X₁=NH, X₂=NH, R₂=Ph, R₃=Br) (60).3-Bromo-5-aminomethyl-4,5-dihydroisoazole (20 mg, 0.11 mmol) and phenylisocyanate (13 uL, 1.0 eq) were dissolved in the mixture of THF (0.5 mL)and DMF (0.1 mL). After 30 min of stirring, the mixture was diluted withethyl acetate and washed with brine. The organic layer was dried overNa₂SO₄ and the solvents were removed by evaporation. The residue waspurified by SiO₂ chromatography to give the title compound.

¹H NMR (acetone-d₆, 400 MHz): δ=8.02 (br, 1H), 7.50 (d, 2H), 7.25-7.20(m, 2H), 6.93 (t, 1H), 6.24 (br, 1H), 4.90-4.86 (m, 1H), 3.56-3.54 (m,2H), 3.48-3.41 (m, 1H), 3.19-3.13 (m, 1H)

MS (ESI): m/z=298.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea(With reference to Formula 5, X₁=NH, X₂=NH,R₂=2-chloro-5-trifluoromethyl-phenyl, R₃=Br) (61). The title compoundwas prepared according to the procedure for compound 60 except using2-chloro-5-trifluoromethyl-phenylisocyanate

¹H NMR (acetone-d₆, 400 MHz): δ=8.82 (s, 1H), 8.12 (br, 1H), 7.62 (d,1H, J=8.0 Hz), 7.30 (d, 1H, J=8.0 Hz), 6.90 (br, 1H), 4.93-4.87 (m, 1H),3.57-3.54 (m, 2H), 3.49-3.42 (m, 1H), 3.20-3.14 (m, 1H)

MS (ESI): m/z=400.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-chloro-2-trifluoromethyl-phenyl)-urea(With reference to Formula 5, X₁=NH, X₂=NH,R₂=4-chloro-2-trifluoromethyl-phenyl, R₃=Br) (62). The title compoundwas prepared according to the procedure for compound 60 except using4-chloro-2-trifluoromethyl-phenylisocyanate.

¹H NMR (acetone-d₆, 400 MHz): δ=8.20 (d, 1H, J=7.6 Hz), 7.66 (br, 1H),7.62-7.60 (m, 2H), 6.82 (br, 1H), 4.89-4.85 (m, 1H), 3.55-3.51 (m, 2H),3.47-3.40 (m, 1H), 3.18-3.12 (m, 1H)

MS (ESI): m/z=400.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-fluoro-phenyl)-urea(With reference to Formula 5, X₁=NH, X₂=NH, R₂=4-fluoro-phenyl, R₃=Br)(63). The title compound was prepared according to the procedure forcompound 60 except using 4-fluoro-phenylisocyanate.

¹H NMR (acetone-d₆, 200 MHz): δ=8.06 (br, 1H), 7.47-7.40 (m, 2H),6.99-6.90 (m, 2H), 5.94 (br, 1H), 4.82-4.76 (m, 1H), 3.45-3.30 (m, 3H),3.17-3.04 (m, 1H)

MS (ESI): m/z=316.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2,5-dimethyl-phenyl)-urea(With reference to Formula 5, X₁=NH, X₂=NH, R₂=2,5-dimethyl-phenyl,R₃=Br) (64). The title compound was prepared according to the procedurefor compound 60 except using 2,5-dimethyl-phenylisocyanate.

MS (ESI): m/z=326.0 [M+H]⁺

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-fluoro-phenyl)-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 1c). ₁H NMR (400MHz, CDCl₃) δ 7.38-7.29 (m, 5H), 7.15-7.11 (m, 2H), 6.99-6.95 (m, 2H),6.30-6.24 (m, 1H), 5.20-5.15 (m, 1H), 5.11-5.07 (m, 2H), 4.73-4.67 (m,1H), 4.38-4.34 (m, 1H), 3.51-3.46 (m, 2H), 3.25-3.15 (m, 1H), 3.09-2.99(m, 2H), 2.95-2.80 (m, 1H); MS (ESI) m/z 500.2 [M+Na]₊.

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(3-fluoro-phenyl)-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 1d).

₁H NMR (400 MHz, CDCl₃) δ 7.35-7.22 (m, 6H), 6.96-6.88 (m, 3H),6.33-6.26 (m, 1H), 5.24-5.17 (m, 1H), 5.11-5.04 (m, 2H), 4.72-4.64 (m,1H), 4.41-4.34 (m, 1H), 3.50-3.43 (m, 2H), 3.26-3.16 (m, 1H), 3.10-3.03(m, 1H), 2.94-2.81 (m, 1H); MS (ESI) m/z 500.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl}carbamicacid benzyl ester (With reference to Table 1, structure 1e). ₁H NMR (400MHz, CDCl₃) δ 8.14 (br, 1H), 7.70-7.63 (m, 1H), 7.37-7.31 (m, 6H),7.22-7.18 (m, 1H), 7.13-7.09 (m, 1H), 7.04-7.02 (m, 1H), 6.15-6.10 (m,1H), 5.45-5.39 (m, 1H), 5.14-5.06 (m, 2H), 4.59-4.47 (m, 2H), 3.40-3.31(m, 3H), 3.20-3.14 (m, 1H), 3.11-3.04 (m, 1H), 2.82-2.74 (m, 1H); MS(ESI): m/z 499.0 [M+H]+, 521.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-hydroxy-1H-indol-3-yl)-ethyl}-carbamicacid benzyl ester (With reference to Table 1, structure 1f). ₁H NMR (400MHz, CDCl₃) δ 7.98 (s, 1H), 7.36-7.33 (m, 5H), 7.21 (d, 1H, J=8.6), 7.06(s, 1H), 7.00-6.99 (m, 1H), 6.79 (d, 1H, J=8.6), 6.30-6.20 (m, 1H),5.57-5.48 (m, 1H), 5.12-5.07 (m, 2H), 4.60-4.44 (m, 2H), 3.43-3.24 (m,3H), 3.11-3.04 (m, 1H), 2.79-2.71 (m, 1H).

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid pyridin-4-ylmethyl ester (With reference to Table 1, structure 3b).₁H NMR (400 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.49 (d, 2H, J=4.7), 8.33-8.27(m, 1H), 7.64-7.61 (m, 1H), 7.13 (d, 2H, J=4.7), 7.06 (d, 2H, J=8.0),6.65 (d, 2H, J=8.0), 5.04-4.93 (m, 2H), 4.70-4.67 (m, 1H), 4.19-4.13 (m,1H), 3.33-3.19 (m, 3H), 3.03-2.93 (m, 1H), 2.86-2.82 (m, 1H), 2.65-2.60(m, 1H); MS (ESI) m/z 477.2 [M+H]+, 499.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid pyridin-3-ylmethyl ester (With reference to Table 1, structure 3c).₁H NMR (400 MHz, DMSO-d₆) δ 9.17 (s, 1H), 8.50-8.48 (m, 2H), 8.29-8.26(m, 1H), 7.62 (d, 1H, J=7.6), 7.51-7.47 (m, 1H), 7.37-7.34 (m, 1H), 7.03(d, 2H, J=8.0), 6.62 (d, 2H, J=8.0), 4.97 (s, 2H), 4.68-4.63 (m, 1H),4.14-4.09 (m, 1H), 3.31-3.26 (m, 3H), 3.02-2.91 (m, 1H), 2.80-2.77 (m,1H), 2.63-2.57 (m, 1H); MS (ESI) m/z 477.2 [M+H]+, 499.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid phenethyl ester (With reference to Table 1, structure 3d). ₁H NMR(400 MHz, DMSO-d₆) δ 9.17 (s, 1H), 8.22-8.19 (m, 1H), 7.29-7.25 (m, 3H),7.21-7.18 (m, 3H), 7.03 (d, 2H, J=8.1), 6.63 (d, 2H, J=8.1), 4.69-4.64(m, 1H), 4.11-4.00 (m, 3H), 3.30-3.18 (m, 3H), 3.03-2.92 (m, 1H),2.80-2.72 (m, 3H), 2.64-2.58 (m, 1H); MS (ESI) m/z 490.2 [M+H]+, 512.2[M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid naphthalen-2-ylmethyl ester (With reference to Table 1, structure3e). ₁H NMR (300 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.29-8.26 (m, 1H),7.89-7.84 (m, 3H), 7.79 (s, 1H), 7.52-7.49 (m, 3H), 7.38 (d, ₁H, J=8.4),7.06 (d, 2H, J=8.1), 6.63 (d, 2H, J=8.1), 5.11 (s, 2H), 4.70-4.64 (m,1H), 4.18-4.12 (m, 1H), 3.29-3.15 (m, 3H), 3.05-2.91 (m, 1H), 2.83-2.79(m, 1H), 2.67-2.60 (m, 1H); MS (ESI) m/z 548.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid 1,1-dioxo-1H-1λ₆-benzo[b]thiophen-2-ylmethyl ester (With referenceto Table 1, structure 3f). ₁H NMR (400 MHz, CDCl₃) δ 7.71 (d, 1H,J=7.2), 7.59-7.56 (m, 1H), 7.54-7.50 (m, 1H), 7.39 (d, 1H, J=7.2), 7.06(s, 1H), 7.03 (d, 2H, J=7.6), 6.83-6.77 (m, 3H), 5.78-5.72 (m, 1H),5.18-5.00 (m, 2H), 4.73-4.66 (m, 1H), 4.38-4.33 (m, 1H), 3.48-3.41 (m,2H), 3.19-3.12 (m, 1H), 3.00-2.88 (m, 3H); MS (ESI) m/z 586.2 [M+Na]₊.

{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-hydroxy-1H-indol-3-yl)-ethyl}-carbamicacid 1,1-dioxo-1H-1λ₆-benzo[b]thiophen-2-ylmethyl ester (With referenceto Table 1, structure 4). ₁H NMR (200 MHz, DMSO-d₆) δ 10.50 (s, 1H),8.57 (s, 1H), 8.26-8.23 (m, 1H), 7.86 (d, 1H, J=7.2), 7.80-7.60 (m, 4H),7.36 (s, 1H), 7.12-7.05 (m, 2H), 6.91 (s, 1H), 6.58 (d, 1H, J=8.4), 4.93(s, 2H), 4.66-4.62 (m, 1H), 4.24-4.20 (m, 1H), 3.25-3.11 (m, 3H),3.03-2.77 (m, 3H); MS (ESI) m/z 625.2 [M+Na]₊.

Example 2

Synthesis of dioxoindole containing tTGase Inhibitors

Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide. 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl chloride (0.10g, 0.41 mmol), prepared by the reaction of the sodium salt of5-isatinsulfonic acid with POCl₃, was dissolved in 5 mL THF. Thissolution was cooled in an ice bath and DIEA (0.14 mL, 2.0 eq) was addedslowly, followed by n-propylamine (35 uL, 1.0 eq). Stirring wascontinued for 40 min and the solution was diluted with ethyl acetate andwashed with brine. The organic layer was dried over Na₂SO₄ and thesolvent was removed by evaporation. The residue was purified by SiO₂chromatography to give the title compound (65 mg, 60%).

¹H NMR (CD₃CN, 400 MHz): δ=9.17 (br, 1H), 8.02 (d, 1H, J=8.0 Hz), 7.93(s, 1H), 7.13 (d, 1H, J=8.0 Hz), 5.62-5.58 (m, 1H), 2.85-2.80 (m, 2H),1.48-1.42 (m, 2H), 0.85 (t, 3H, J=7.2 Hz)

MS (ESI): m/z=−267.1 [M−H]⁻

Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidbenzylamide. The title compound was prepared from benzyl amine followingthe procedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide.

¹H NMR (CD₃CN, 400 MHz): δ=9.19 (br, 1H), 7.98 (d, 1H, J=8.4 Hz), 7.85(s, 1H), 7.31-7.21 (m, 5H), 7.07 (d, 1H, J=8.4 Hz), 6.11 (t, 1H, J=6.3Hz), 4.11 (d, 2H, J=6.3 Hz)

MS (ESI): m/z=−315.2 [M−H]⁻

Synthesis of (S)-1-(2,3-Dioxo-Z3-dihydro-1H-indole-5-sulfonyl)-pyrrolidine-2-carboxylic acid methylester. The title compound was prepared from L-Pro-OMe following theprocedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide.

¹H NMR (CDCl₃, 200 MHz): δ=8.85 (br, 1H), 8.15-8.11 (m, 2H), 7.11 (d,1H, J=8.8 Hz), 4.47-4.41 (m, 1H), 3.74 (s, 3H), 3.45-3.39 (m, 2H),2.20-1.94 (m, 4H)

MS (ESI): m/z=338.9 [M+H]⁺

Synthesis of(S)-2-(2,3-Diox-2,3-dihydro-1H-indole-5-sulfonylamino)-3-phenyl-propionamide.The title compound was prepared from L-Phe-NH₂ following the procedurefor 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.

¹H NMR (CD₃CN, 200 MHz): δ=10.70 (br, 1H), 7.78 (d, 1H, J=8.4 Hz), 7.64(s, 1H), 7.15-7.06 (m, 6H), 6.90 (d, 1H, J=8.4 Hz), 6.79 (br, 1H), 6.08(br, 1H), 3.98-3.87 (m, 1H), 3.04-2.95 (m, 1H), 2.76-2.64 (m, 1H)

MS (ESI): m/z=−372.2 [M−H]⁻

Synthesis of(S)—N-(2-Dimethylamino-ethyl)-2-(2,3-dioxo-2,3-dihydro-1H-indole-5-sulfonylamino)-3-phenyl-propionamide. The title compound was prepared fromL-Phe-NHCH₂CH₂NMe₂ following the procedure for2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.

¹H NMR (CD₃CN, 400 MHz): δ=7.84 (d, 1H, J=8.0 Hz), 7.69 (s, 1H),7.22-7.12 (m, 6H), 6.98 (d, 1H, J=8.0 Hz), 6.76 (br, 1H), 3.96-3.93 (m,1H), 3.10-3.02 (m, 2H), 3.00-2.95 (m, 1H), 2.78-2.72 (m, 1H), 2.22-2.17(m, 2H), 2.15 (s, 6H)

MS (ESI): m/z=445.2 [M+H]⁺

Synthesis of 6-Bromo-7-methyl-1H-indole-2,3-dione. Chloral alcoholate(0.43 g, 1.05 eq) and Na₂SO₄ (2.84 g, 20 mmol) were dissolved in 10 mLwater. 3-Bromo-2-methylaniline (0.33 g, 1.77 mmol) was added to thesolution followed by 0.16 mL conc. HCl aqueous solution and NH₂OH.HCl(0.38 g, 3.0 eq). The mixture was refluxed for 15 min and stirring wascontinued for additional 1 hr at RT. The precipitate was collected byfiltration, washed with water and dried under vacuum. This precipitatewas dissolved in 1 mL H₂SO₄ and the solution was heated (80° C.) for 15min. After cooling down to RT, the mixture was poured into ice-watermixture and the precipitate was collected, washed with water and driedunder vacuum to give the title compound (0.26 g, 61%).

¹H NMR (CD₃CN, 200 MHz): δ=9.02 (BR, 1h), 7.38 (d, 1H, J=7.8 Hz), 7.30(d, 1H, J=7.8 Hz), 2.30 (s, 3H)

MS (ESI): m/z=−238.2 [M−H]⁻

Synthesis of 7-Methyl-6-phenyl-1H-indole-2,3-dione.6-Bromo-7-methyl-1H-indole-2,3-dione (100 mg, 0.38 mmol) andphenylboronic acid (53 mg, 1.1 eq) were dissolved in 10 mL DME.Pd(PPh₃)₄ (22 mg, 0.05 eq) were added followed by NaHCO₃ (65 mg, 2.0 eq)dissolved in 10 mL water. The mixture was refluxed for 2.5 hr and theorganic solvent was removed by evaporation: The mixture was extractedwith ethyl acetate and the combined organic layers were dried overNa₂SO₄ and purified by SiO₂ chromatography to give the title compound(50 mg, 51%).

¹H NMR (CDCl₃, 400 MHz): δ=8.53 (br, 1H), 7.52 (d, 1H, J=7.6 Hz),7.49-7.43 (m, 3H), 7.31 (d, 2H, J=6.4 Hz), 7.03 (d, 1H, J=7.6 Hz), 2.16(s, 3H)

MS (ESI): m/z=−236.3 [M−H]⁻

Inhibition of tTG. tTG (9 μM) was inactivated in 200 mM MOPS, pH=7.1, 5mM CaCl₂, 1 mM ETDA at 30° C. containing 0-600 μMPro-Gln-Pro-Aci-Leu-Pro-Tyr. Every 20 minutes a 40 μl aliquot wasremoved and residual tTG activity was assayed in 0.5 ml reactioncontaining 200 mM MOPS, pH=7.1, 5 mM CaCl₂, 1 mM ETDA, 10 mMα-ketoglutarate, 180 U/ml glutamate dehydrogenase (Biozyme laboratories)at 30° C. for 20 minutes by measuring the decrease of absorption at 340nm. Residual activity was corrected by the corresponding uninhibited tTGreaction (0 μM inhibitor) and fitted to an exponential decay. Kineticparameters were obtained by double-reciprocal plotting of the apparentsecond-order inactivation constant or, for isatin analogs, by fittingthe data for reversible inhibitors to a standard Michaelis Mentenequation with a competitive inhibition constant. The results of theseinhibition experiments are shown in below.

TABLE 2 Tissue transglutaminase inhibition by dihydroisoxazolesk_(inh)/K_(I) Tested Compound K_(I) (M) K_(inh) (min⁻¹) (min⁻¹M⁻¹)1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-urea 1.1 × 10⁻³0.89 810 1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2-chloro-5- 0.91× 10⁻³  0.95 1000 trifluoromethyl-phenyl)-urea1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-chloro-2- 1.3 × 10⁻³1.1 850 trifluoromethyl-phenyl)-urea1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-fluoro- 1.3 × 10⁻³ 1.0770 phenyl)-urea1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2,5-dimethyl- 0.96 ×10⁻³  0.97 1000 phenyl)-urea

TABLE 3 Tissue transglutaminase inhibition by Istatin derivatives 7

Tested compound (R) K_(l) (M) R1 = R2 = R3 = H 8.6 × 10⁻⁴ R2 = R3 = H,R1 = NO₂ 4.8 × 10⁻⁵ R2 = R3 = H; R1 = I 2.2 × 10⁻⁵ R2 = R3 = H; R1 = F1.8 × 10⁻⁵ R1 = R2 = H; R3 = Ph   4 × 10⁻⁴ R1 = R3 = H; R2 = Ph 3.5 ×10⁻⁴

The above results demonstrate that the compounds tested have tTGaseinhibitory activity.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A tTGase inhibitor of the formula:

wherein Ri and R2 are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups, an amino acid, a peptide, a peptidomimetic, ora peptidic protecting group; wherein R2 can additionally be selectedfrom the group consisting of LPYPQPQLPY, LPFPQPQLPF-NH₂, LPYPQPQLP,LPYPQPQLPYPQPQPF, where X2-15 is a peptide consisting of any 2-15 aminoacid residues followed by a C-terminal proline; R3 is selected from F,and Br; n is from 0 to 10; and X is selected from the group consistingof O and NH, other than acid benzyl ester.
 2. The inhibitor of claim 1,wherein Ri is selected from the group consisting of BnO, Me, Cbz, Fmoc,Boc, PQP, PQPQLPYPQP, QLQPFPQP, LQLQPFPQPLPYPQP, where X2-15 is apeptide consisting of any 2-15 amino acid residues followed by aN-terminal proline.
 3. The inhibitor of claim 1, wherein R2 is selectedfrom the group consisting of hydrohy-phenyl)-methyl, OMe, OtBu, LPY,LPF-NH₂.
 4. The inhibitor of claim 1, wherein is Br.
 5. The inhibitor ofclaim 1, wherein said tTGase inhibitor is selected from the groupconsisting of:{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester; 5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-butyricacid methyl ester; (S)-2-Benzyloxycarbonylamino-acid methyl ester;(S)-2-ester; acid benzyl ester; propionamide; acid benzyl ester; acidbenzyl ester; [(S)-1-[(3-Bromo-4,5-acid benzyl ester; carbamic acidbenzyl ester; acid benzyl ester;5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-urea;chloro-5-trifluoromethyl-phenyl)-urea;chloro-2-trifluoromethyl-phenyl)-urea; fluoro-phenyl)-urea; urea;{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-fluoro-phenyl)-ethyl}-carbamicacid benzyl ester;{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-acid benzyl ester;5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl}-carbamicacid benzyl ester;{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylamethyl)-carbamoyl]-2-(5-hydroxy-1H-indol-3-yl)-ethyl}-carbamicacid benzyl ester; acid pyridin-4-ylmethyl ester;{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl}-carbamicacid ester; acid phenethyl ester; 5-dihydro-ester;{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-[b]thiophen-2-ylmethylester; Bromo-4, carbamic acid 1, [b]thiophen-2-ylmethyl ester.
 6. AtTGase inhibitor of the formula:

where R2 and R3 are independently selected from H, a halo group, alkyl,aryl, and NO2.
 7. The tTGase inhibitor of claim 11, wherein saidinhibitor is selected from the group consisting of: 2,3-Dioxo-2, acidpropylamide; 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidbenzylamide;(S)-1-(2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl)-pyrrolidine-2-carboxylicacid methyl ester; (S)-2-(2,3-Dioxo-2,3-dihydro-1H-indole-5-;3-dioxo-2,3-; 2,3-dione; 3-dione
 8. A formulation for use in treatmentof Celiac Sprue and/or dermatitis herpetiformis, comprising: aneffective dose of the tTGase inhibitor according to any of claims 1-7and a pharmaceutical acceptable excipient.
 8. A formulation for use intreatment of Celiac Sprue and/or dermatitis herpetiformis, comprising:an effective dose of the tTGase inhibitor according to any of claim 1and a pharmaceutically acceptable excipient.
 9. A method of treatingCeliac Sprue and/or dermatitis herpetiformis, the method comprising:administering to a patient an effective dose of a formulation accordingto claim 8; wherein said tTGase inhibitor attenuates gluten toxicity insaid patient
 10. The method of claim 9, wherein said formulation isadministered with a glutenase.
 11. The method according to claim 9,wherein said formulation is administered orally.
 12. The methodaccording to claim 9, wherein said formulation comprises an entericcoating.